As an approach for realizing a more advanced generation DNA sequencer, techniques using nanopores have been studied. That is, an aperture (nanopore) having a size similar to DNA are provided in a thin film membrane, an upper and lower chambers of the thin film membrane are filled with an aqueous solution, electrodes are provided in the two chambers so as to be in contact with the aqueous solution, DNA to be measured is put in one of the chambers, a potential difference is caused between the electrodes provided in the two chambers to subject the DNA to electrophoresis to allow the DNA to pass through the nanopore, and during this time, the temporal change in the ionic current flowing between the two electrodes is measured, thereby determining a structural feature or the basic sequence of the DNA. The above technique is useful for acquiring a structural feature of not only DNA but also various biological molecules.
For producing the nanopore device, methods using a semiconductor substrate, a semiconductor material, or a semiconductor process, which has a high mechanical strength or other characteristics, are attracting attentions. For example, a thin film membrane can be formed with a silicon nitride film (SiN film). Voltage stress is applied to a membrane in an ionic aqueous solution to cause dielectric breakdown, whereby fine pinholes can be bored to form a nanopore in the membrane (NPL 1). In an alternative method, a nanopore can be formed by subjecting a membrane to etching by a focused electron ray.
One of important factors for determining accuracy in DNA reading by a nanopore sequencer is a film thickness of the membrane. Specifically, a smaller thickness of the membrane is more preferred. The reason is that the interval between adjacent two of four kinds of bases arranged in a DNA strand is approximately 0.34 nm, and the larger the thickness of the membrane is as compared to the interval, the larger number of bases are simultaneously placed in one nanopore. In this situation, a signal obtained by measuring a current is a signal generated by multiple bases, resulting in reduction of the accuracy of base sequence determination and leading to a complicated signal analysis. Also when acquisition of structural features is intended for various biological molecules other than DNA, a larger thickness of the membrane results in reduction of space resolution. Accordingly, for enhancement of accuracy in structure determination of a substance to be measured, it is highly important to make the thickness of the membrane having a nanopore as small as possible.
In order to reduce the thickness of a membrane, it is obviously preferred that the membrane area is as small as possible. The smaller the membrane area, the lower the possibility of presence of unavoidable defects (weak spots or pinholes due to bonding failure between atoms) generated during forming the membrane in the membrane. In addition, when the membrane is formed, it is important to avoid a process involving the possibility of scratching and breaking the membrane as much as possible.
Hereinunder, attempts of reduction in thickness of a membrane will be described with explanation of some examples of a conventional representative method for forming a membrane using a semiconductor material.
In the simplest method of forming a membrane, a film of a material for the membrane (e.g., SiN) is formed on a Si substrate, a film of SiN is formed on the back surface, a part of the SiN film on the back surface is etched so as to expose the Si substrate, and then the Si substrate is etched with an aqueous solution of KOH or TMAH from the part of the back surface where the Si is exposed, toward the front surface, whereby a membrane supported on the Si substrate can be formed. In this method, it is difficult to make the area of the membrane small. Although the etching of the Si substrate with the KOH aqueous solution or TMAH aqueous solution is a crystal anisotropy etching in which only the (100) face is preferentially etched, etchings in other directions than the (100) face proceeds in certain degrees and variation in etching shape is also large. In particular, etching proceeds also in an unexpected direction with a crystal defect present in Si as the starting point, and therefore the variation in shape increases. In addition, the thickness of the Si substrate to be etched is generally large and at least 100 um or more (for example, 725 um in the case of a Si wafer of 8 inches). The thickness of the substrate varies in one wafer or among wafers, and generally, there is a variation of 1 um or more. Thus, a membrane having a size greatly deviated from the membrane size expected from the shape of a mask of the SiN film on the back surface is formed. For the above reason, according to the result of our previous study, it has been difficult to stably form a membrane having an area of 50 um×50 um square or smaller size by this production method. However, in order to aim at forming a thin film membrane, it is required to make the area of the membrane further smaller.
In another production method, as described in NPL 1, a SiN film is formed on a Si substrate, then a SiO2 film is formed on the SiN film, then SiN films are formed on the SiO2 and the back surface of the Si substrate, then a part of the uppermost SiN film on the front surface side of the wafer is patterned by dry-etching to expose the SiO2 film under the SiN film, then a part of the SiN film on the back surface is etched so as to expose the Si substrate, the Si substrate is etched from the back surface with a TMAH aqueous solution, then remove the SiO2 film on the SiN film with an HF aqueous solution, whereby a SiN membrane can be formed. According to this method, if a hole pattern of, for example, 100 nm square or smaller is formed by using a latest lithography technique and dry etching in the patterning of the SiN on the SiO2, after the subsequent SiO2 etching with the HF aqueous solution, the thinnest area of the membrane (an area with a single layer of the SiN membrane) can be made into a size of approximately 100 to 500 nm square inclusive of the variation. In this respect, the method is advantageous in reduction of the membrane thickness.
However, an HF aqueous solution etches a SiN film although the etching rate is lower as compared with the case of a SiO2 film. For this reason, the contact of the HF aqueous solution with the SiN membrane of the thin film part causes breaking of the membrane. In the results of our experiments, the lower limit of the thickness of the membrane in the thinnest part is 7 nm in the above method.
In another method, as described in NPL 2, a thick SiN membrane is formed, and the thickness is reduced by dry etching in a partial area thereof. According to this method, since the thickness of the membrane can be reduced only in an area of a limited part by a latest lithography technique and dry etching, it is possible to achieve the surface area reduction in a thin film membrane part. However, variation in the dry etching rate among different batches is large, and the variation in the etching rate in one wafer surface is also large. Furthermore, since the SiN film before etching has a large thickness, variation in the initial film thickness is also large. For this reason, the thickness of the obtained membrane has large variations among different batches and different samples, relative to the target thickness. In dry etching, ions with a high energy collide with the membrane and therefore damage the membrane. In an ultrathin film area, therefore, there is a possibility of breaking the membrane, and hence the method is unsuitable to reduction of the membrane thickness. According to the description in NPL 2, the thickness of the SiN film is 5 to 8 nm.
Incidentally, a material that is used most widely as a membrane material is SiN. SiN has a high density, is hydrophilic, and is highly excellent in chemical stability. These give a great advantage for a nonopore sensor used in an aqueous solution. In fact, there are many studies in which a nanopore was formed in a SiN membrane and DNA passing through the nanopore was confirmed, other than NPL 1 and NPL 2, and a highly stable passing of DNA through a nanopore was confirmed. Thus, SiN is one of materials that are currently used most frequently as a membrane material for a nanopore sensor. In addition, SiN is excellent in mechanical strength and the membrane is difficult to break. SiN is one of the most general materials that are used in a semiconductor process, and is advantageous also in a very high compatibility with a conventional semiconductor process (for example, CMOS process). That is, a membrane of SiN can be produced in most semiconductor lines in the world. For this reason, a sensor (nanopore sensor) with a SiN membrane is expected to be spread to the industry without any large barrier. Even in the case where an MOS transistor circuit for measurement is installed on the same circuit board with a nanopore sensor, the membrane made of a SiN material is not a large barrier. For the above reasons, SiN is being greatly expected as a material of a nanopore membrane having a very small thickness.